The native peptide α-melanocyte-stimulating hormone (α-MSH) is known as the native agonist for the type 1, the type 3, the type 4 and the type 5 melanocortin (MC) receptor. The MC receptors belong to the class of G-protein coupled receptors. All receptor subtypes are coupled to a G-stimulatory protein, which means that receptor stimulation involves increased production of cAMP. ACTH is the native ligand to the Type 2 receptor (MC2).
A series of studies have been performed on the MC receptors in a variety of tissues. Type 1 receptor (MC1), to which α-MSH binds with great affinity, is known to be expressed in several tissues and cells such a brain, including astrocytes, testis, ovary, macrophages and neutrophils. MC1 is likely to be expressed, however, in an even wider range of tissues although this remains to be established. The selectivity for the MC receptors to bind different MSH peptides varies. MC1 binds with great affinity α-MSH and with lower affinity also β-MSH, γ-MSH and ACTH. MC2 has been reported only to bind ACTH, but none of the MSH peptides. The highest affinity for the ligands of the other receptors include γ-MSH (MC3-receptor), and β-MSH (MC4-receptor). In contrast, MC5 binds with much lower affinity the MSH peptides with the same pattern as MC1 (i.e. highest affinity for α-MSH).
MSH-peptides acting through stimulation of the MC-receptors have a variety of functions including immunomodulation, anti-inflammation, body temperature regulation, pain perception, aldosterone synthesis, blood pressure regulation, heart rate, vascular tone, brain blood flow, nerve growth, placental development, synthesis/release of a variety of hormones such as aldosterone, thyroxin, prolactin, FSH. ACTH has a major effect on stimulating steroidoneogenesis. Also α-MSH induces pigment formation in skin.
It is important to emphasize that a number of actions of MSH peptides, especially α-MSH, are not fully established with respect to which receptors are involved. The anti-inflammatory action of α-MSH has been speculated to involve a variety of processes including interference with NO production, endothelin-1 action, interleukin 10 formation, which again is linked to MC1 receptors expressed in macrophages and monocytes.
MC receptor stimulation with α-MSH has been shown to be important in a variety of inflammatory processes (Lipton and Catania 1997): 1) Inhibit chemotactive migration of neutrophils (Catania 1996). 2) α-MSH including analogs inhibit the release of cytokine (IL-1, TNF-α) in response to LPS treatment (Goninard 1996). 3) Inhibit TNF-α in response to bacterial endotoxin (Wong, K. Y. et al., 1997). 4) ICV or IP administration of α-MSH inhibit central TNF-α production by locally administered LPS. 5) α-MSH has been shown to reduce the inflammation in experimental inflammatory bowel disease (Rajora, N. et al., 1997), ishemia-induced acute renal failure (Star, R. A. et al., 1995). 6) α-MSH also have some protective effect by inhibiting the induction and elicitation of contact hypersensitivity and induces hapten tolerance, and it is speculated that α-MSH may mediate important negative regulation of cutaneous inflammation and hyper-proliferative skin diseases (Luger, T. A., 1997). To this end α-MSH causes increased IL-8 release from dermal microvasculature endothelial cells (Hartmeyer, M., 1997).
Both hypoxia (ischemia) and reperfusion injuries are important factors in human pathophysiology. Examples of tissue hypoxia that predispose to injury during reperfusion include circulatory shock, myocardial ischemia, stroke, temporary renal ischemia, major surgery and organ-transplantation. Because diseases due to ischemia are exceedingly common causes of morbidity and mortality and because organ transplantation is increasingly frequent, treatment strategies with the potential of limiting reperfusion injuries is of great need in order to improve public health. The underlying pathophysiology of ischemia reperfusion injuries is complex and involves not only a classical inflammatory reperfusion response with neutrophil-infiltration, but also cytokine gene expression including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, interferon-γ, and intercellular adhesion molecule-1 (ICAM-1) within the reperfusion tissue/organ. Furthermore, it has been suggested that locally produced TNF-α contributes to postischemic organ dysfunction as in the postinfarctional heart by direct depression of contractility and induction of apoptosis. Because of the complex nature of ischemia and/or reperfusion injuries simple anti-inflammatory treatment concepts have been shown ineffective: Most experimental studies therefore point to the fact that concomitant interaction with more than one of the activated pathways is needed in order to protect against reperfusion injuries. α-MSH have been shown to have both anti-inflammatory, anti-oxidative and anti-apoptotic abilities, which gives a good explanation for the effectiveness of this compound in order to protect against reperfusion injuries.
It is known that certain modifications of amino acid residues in the α-MSH amino acid sequence result in an increased receptor affinity (for e.g. the MC4 receptor), prolonged biological activity or a more receptor-specific binding profile of the peptide (Schiöth et al. 1998, Hruby et al. 1995, Sawyer et al. 1980, Hiltz et al. 1991, Scardenings et al. 2000). However, when aiming towards generation of peptidic drugs, these peptides still have problems with low stability towards enzymatic degradation.
As stated above, the problem in the development of peptidic therapeutical active drugs is that peptides are rapidly and very effectively degraded by enzymes, generally with half-lives in the range of minutes. Proteases and other proteolytic enzymes are ubiquitous, particularly in the gastro-intestinal tract, and therefore peptides are usually susceptible to degradation in multiple sites upon oral administration, and to some extent in the blood, the liver, the kidney, and the vascular endothelia. Furthermore, a given peptide is usually susceptible to degradation at more than one linkage within the backbone; each locus of hydrolysis is mediated by a certain protease. Even if such obstacles are overcome, for neuropeptides in particular, difficulties have been encountered in their transport across the blood-brain barrier.
In order to increase the metabolic stability of peptides, a technology called SIP (Structural Induced Probe) has been developed by Larsen et al. 1999 (WO 99/46283). The SIP technology is based on the use of structure inducing probes, which are represented by short peptide sequences, i.e. (Lys)6 added to the C-terminal or to the N-terminal or both the C- and N-termini of the parent peptide. The structural inducing probe constrains the parent peptide into a more ordered conformation based on intramolecular hydrogen bonds, whereby the peptide chimer (peptide linked to the probe) is less susceptible to proteases in contrast to peptides in the random-coil conformation. As a result of the structuring, the peptide chimer is much more difficult for a protease to degrade. The addition of a SIP to a biologically active peptide generally results in an increase in the enzymatic stability of the peptide while the biological activity at the same time is maintained (Rizzi et al. 2002).